The glucose oxidase method of assaying for glucose used by Trinder is well-known in the art. Such method uses reagents, which comprise glucose oxidase, a substance having peroxidase activity, 4-aminoantipyrine and a phenolic or naphtholic coupler. The method and reagents are described in detail in the following two references: Trinder, P., Ann. Clin. Biochem., 6 (1969), 24, and Trinder, P., J, Clin, Pathol., 22 (1969), 246.
Sodium fluoride is used as a preservative in blood and blood serum samples which are to be held for an extended period of time, i.e. up to 10 days (Henry, Clinical Chemistry, 2 Ed. p. 385). Such a preservative is routinely used at a level of 250 mg/dl of blood or blood serum. The addition of 250 mg/dl of sodium fluoride to blood serium has been found to decrease the apparent glucose concentration, as measured utilizing multilayer elements of the type described in U.S. Pat. No. 3,992,158, issued November 16, 1976, by 20-30 mg/dl at a concentration of 200 mg glucose/dl. This problem is compounded by the following factors: 1) the preservative is not always used and 2) when the preservative is incorporated within blood withdrawal apparatus such as found in commercial embodiments, the concentration of preservative is known only when the container is filled to capacity. Since often the containers are not filled to capacity (due to loss of vacuum) the sodium fluoride concentration may increase as much as 2-3 fold over the 250 mg/dl level.
In view of the variability in sodium fluoride concentration which may be encountered, it is clear that a need exists for a multilayer element for the analysis of glucose which exhibits reduced sensitivity to sodium fluoride.
Although the concentrations of fluoride ion used for this purpose have apparently not caused noticeable interference with prior-art methods for analyzing for glucose probably due to dilution techniques commonly used in prior art solution quantitative assays and the semi-quantitative character of prior art "dry" methods for glucose detection, it has been found that, when a Trinder's reagent system as described above is incorporated into a multilayer element of the type described in U.S. Pat. No. 3,992,158, the fluoride ion used as a preservative contributes a substantial negative bias. The presence and elimination of this bias using the method described herein will be shown in the examples below.
U.S. Pat. No. 3,992,158 issued Nov. 16, 1976, to Przybylowicz and Millikan, describes integral multilayer elements for the detection of various biological materials.
Example 3 of U.S. Pat. No. 3,992,158 describes an element for the detection of glucose using glucose oxidase, peroxidase and an indicator system of the type described herein in an element similar to that described in the instant application. However, no pH is specified for the reagent layer containing the foregoing reagent system. A layer prepared according to this example would have a pH of above about 7.0.
U.S. Pat. No. 3,983,005 issued Sept. 28, 1976, to Goodhue et al, describes in Example 3 the use of 4-aminoantipyrine and a naphtholic coupler for the detection of hydrogen peroxide produced on contact with cholesterol oxidase in an element of the type described herein. According to this example, the indicator reaction is carried out at a pH of 7.0.
U.S. Pat. No. 3,886,045 issued May 27, 1975, describes a test composition for glucose assay comprising glucose oxidase, peroxidase, sodium or potassium ferrocyanide, an aminoantipyrine and a phenolic coupler. It is disclosed that such composition, always including the ferricyanide, are useful when buffered at a pH of between about 5.5 and 8.0. There is no disclosure that compositions which omit the ferrocyanide are useful throughout this pH range. This patent also includes an extensive discussion of phenols useful in analytical reagents of the type described herein.